MOLECULAR STUDIES OF THE BIOLOGICAL AND CATALVTIC ACTIVITIES OF AIBONUCLEASES by Peter A. Leland A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Biochemistry) at the UNIVERSITY OF WISCONSIN-MADISON 2000 ....r .... A dissertation entitled ....;­ .... Molecular Studies of the Biological and ~ Catalytic Activities of Ribonucleases submitted to the Graduate School of the University of Wisconsin-Madison in partial fulfillment of the requirements for the degree of Doctor of Philosophy by Peter Andrew Leland Date of Final Oral Examination: May 30, 2000 Month & Year Degree to be awarded: December May August 2000 * * • * * • * * * * • * * * * • * * * * * * • * * • • • * * * • * * * * * * * • * * • * * • * * * • * * * * Signature, Dean of Graduate School r. Abstract Bovine pancreatic ribonuclease A (RNase A) catalyzes cleavage of RNA. Homologs of RNase A effect diverse biological phenomena. Onconase™ (ONC). an amphibian ribonuclease. is a potent toxin to cancer cells. Angiogenin (ANG). a ribonuclease present in human plasma. promotes angiogenesis. The cytotoxic activity of ONC and the angiogenic activity of ANG depend on the enzymes' ribonucleolytic activity. This Dissertation describes factors that alter the ribonucleolytic activities. and consequently. the biological activities of ONC and ANG. Chapters Two and Three show that Ribonuclease Inhibitor (R!). a ubiquitous cytosolic protein. distinguishes ONC and other cytotoxic ribonucleases from their nontoxic homologs. Chapter Four shows that high conformational stability helps to preserve the catalytic activity of ONC within the cytosol and thereby contributes to its cytotoxicity. Chapter Five describes solution conditions. including pH and [Na+]. that affect the catalytic activity of ANG. Definition of the factors that influence ribonucleolytic activity offers insight into the molecular mechanisms ribonuclease biology. Cytotoxic ribonucleases enter the cytosol, where they degrade RNA and cause cell death. RI binds to members of the RNase A superfamily with inhibition constants that span 10 orders of magnitude. RI plays an integral role in defining ribonuclease cytotoxicity. RNase A is not cytotoxic and binds RI with high affinity. ONC. in contrast. binds RI with low affinity. To disrupt the RI - RNase A interaction. RNase A residues that contact RI were replaced with arginine or aspartate. Replacing Gly88 with arginine or aspartate yields variants of ii R.Nase A that retain catalytic activity in the presence of RI and are cytotoxic to a human cancer grown in culture. Like RNase A. human pancreatic ribonuclease can be made cytotoxic by specifically decreasing its susceptibility to RI. ERDD hpRNase. which is the L86E1N88R1G89D1R91D variant. is shown to have 103 -fold less affinity for RI than does wild-type hpRNase. Moreover. ERDD hpRNase is toxic to a human cancer grown in culture. Replacing Arg4 and Val I 18 of ERDD hpRNase with cysteine residues to form a fifth disulfide bond increases conformational stability. decreases affinity for RI. and (most significantly) increases cytotoxicity of the ERDD hpRNase variant. Because ERDD hpRNase is derived from a human protein. it is unlikely to cause an immune reaction if used as a cancer chemotherapeutic. The conformational stability of ONe is remarkable-the midpoint of its thermal denaturation curve is 90 °e. ONe and its amphibian homo logs have a C-terminal disulfide bond. which is absent in RNase A. Replacing this cystine with a pair of alanine residues decreases significantly the conformational stability of ONe. In addition. deletion of the C-tenninal disulfide bond diminishes significantly the cytotoxicity of ONe. The biological activity of ANG is dependent on its ribonucleolytic activity. which is several orders of magnitude lower than that of RNase A. Efficient heterologous production of ANG is achieved by replacing two sequences of rare codons with codons favored by Escherichia coli. Catalysis by ANG is dependent on the length of its substrates. When a substrate is extended from two nucleotides to four or six nucleotides. values of kcalKM increase 5- or 12-fold. respectively. The ANG pH-rate profile is a classic bell-shaped curve. iii with pKI = 5.0 and pKz = 7.0. Finally. the ribonucleolytic activity of ANG is sensitive to salt concentration-a lO-fold decrease in [Na+] causes a l7O-fold increase in the value of kca/KM. Likewise. ANG binding to a tetranucleotide substrate analog is dependent on [Na+]. These data provide a systematic evaluation of substrate binding and catalysis by ANG. iv Acknowledgements Although my name alone appears on the title page. this Dissertation is the product of many talented individuals. The students. technicians. and postdoctoral fellows in the Raines laboratory create an outstanding environment in which to conduct scientific research and are themselves outstanding sources of assistance. In particular. Dr. L. Wayne Schultz helped to design the cytotoxic ribonuclease A variants described in Chapter One. Dr. Byung-Moon Kim helped to create some of the ribonuclease variants described in Chapter Two. Chapter Three. and Chapter Four. Chiwook Park made significant contributions to the analysis of the angiogenin pH-rate and salt-rate profiles presented in Chapter 5. Kristine E. Staniszewski is a gifted undergraduate student. with whom I had the privilege to work with for the past two years. Ms. Staniszewski helped to collect data that appears in Chapter Three and Chapter Four. Tony A. Klink. Kenneth J. Woycechowsky. Marcia C. Haigis. Cara L. Jekins. Bradley R. Kelemen. Richele L. Abel. and Kimberly A. Dickson have willingly served as editors of this Dissertation. Ronald T. Raines has served as an excellent advisor during my tenure in the Raines lab. His efforts guaranteed that the necessary resources - both physical and intellectual- were always available. Finally. I thank my family for continually supporting my education. I must also thank my wife. Jill. Without her constant presence and her laughter, this work would not have been possible. v Table of Contents Abstract ................................................................................................................................. i Acknow ledgements ................................... '" ....................................................................... .iv Table of Contents .................................................................................................................. v List of Figures ................................................................................................................... viii List of Tables ........................................................................................................................ x List of Abbreviations ............................................................................................................ xi Chapter One Introduction ... '" ......................................................................................................... 1 Chapter Two Ribonuclease A Variants with Potent Cytotoxic Activity ......................................... 29 2.1 Abstract ............................................................................................................. 30 2.2 Introduction ....................................................................................................... 31 2.3 Experimental Procedures .................................................................................... 34 2.4 Results ............................................................................................................... 43 2.5 Discussion ......................................................................................................... 48 2.6 Acknowledgements ............................................................................................ 52 Chapter Three Endowing Human Pancreatic Ribonuclease with Cytotoxic Activity ........................ 63 3.1 Abstract ............................................................................................................. 64 3.2 Introduction ....................................................................................................... 65 vi 3.3 Experimental Procedures ................................................................................. '" 67 3.4 Results ............................................................................................................ '" 76 3.5 Discussion ......................................................................................................... 78 3.6 Acknowledgements ............................................................................................ 85 Chapter Four .................................................................................. , .................................... 94 A synapomorphic disulfide bond is critical for the conformational stability and cytotoxicity of an amphibian ribonuclease ............................................................... 94 4.1 Abstract ............................................................................................................. 95 4.2 Introduction ....................................................................................................... 96 4.3 Experimental Procedures .................................................................................... 97 4.4 Results ............................................................................................................